The number of kidney transplants performed every year in the US is increasing. While early graft survival has improved over the past decade, long-term survival has not significantly changed during the same time and remains unsatisfactory. Accumulating evidence suggests that chronic humoral rejection (CHR) is responsible for a large proportion of late kidney graft losses. Seemingly, CHR does not respond well to conventional immunosuppressive therapies. A better understanding of the pathophysiology of this complication will lead to the development of new treatments and reduce the rate of rejection. The long-term goal of our proposed research is to understand the immune mechanisms leading to CHR. CHR is characterized by the development of donor specific antibodies. These antibodies presumably result from a CD4+ T cell dependent B cell response directed to the allograft. Yet, direct evidence of this mechanism is scarce. On the other hand, deficiency in regulatory T cells leads to aberrant B cell activation and autoantibody production in humans. Autoantibodies are prevalent in transplant recipients. It is also known that immunosuppressive drugs used to prevent rejection, directly impair Treg function. We propose that CHR results from Treg deficiency leading to the deregulation of peripheral B cells and concomitant development of allo- and autoantibodies. In a comprehensive analysis, our studies will determine whether Treg deficiency and the co-development of allo- and autoantibodies correlate with CHR in kidney transplant recipients. We will also examine possible mechanisms whereby Treg deficiency induces B cells deregulation in patients with CHR. Experiments to verify our proposed model will be carried out in 3 specific aims: Aim-1. To examine whether CHR is associated with increased autoantibody titers. Utilizing a proteomics array approach, we will identify autoantigenic targets of antibody responses in CHR. We will then assess the co development of autoantibodies to these targets as well as alloantibodies in correlation with the occurrence of CHR. Aim-2. To determine whether CHR correlates with Treg deficiency Phenotypic and molecular assessment of Treg populations in patient samples will determine whether CHR correlates with reduced numbers and frequencies of these cells. In vitro cell based assays will also be used to assess whether CHR correlates with reduced Treg suppressive activity. Aim-3. To define mechanisms of B cell deregulation in CHR Phenotypic and functional assessment of B cell subsets will determine whether deregulation in CHR patients is due to defective B cell development or exacerbated B cell activation in the periphery. In vitro cell based assays will investigate the cellular mechanisms whereby Treg control B cell activation. Lastly, we will examine the possibility that B cells differentiate directly in the graft in patients with CHR.